The overall goal of the proposed research is to investigate metal binding sites in large RNA molecules and their influence on RNA structure, stability, and dynamics. RNA displays a rich array of cellular functions through complex interactions with other nucleic acids and proteins. RNA-metal interactions are critical to both structure and function, and effective, direct probes of the influence of metal sites on the structural biology of RNA are required. In work carried out under this project, direct measurements of metal-binding in RNA systems will be measured using Mn2+ as an EPR-detectable spectroscopic probe. The influence of both Mg2+ and Mn2+ on metal binding, RNA structure, and thermodynamic parameters will be investigated. Insight into the conformational dynamics of RNA will be sought using site-specific, EPR-active nitroxide spin labels. Taken together, these studies will provide important information about RNA structure and stability as a function of solution conditions, and will provide new tools for the investigation of more elaborate and conformationally dynamic RNA complexes.Specific aims of the current proposal include: Investigating the unique 'metal ion core' from the Group I Intron, with a goal of understanding specifically how metal ions contribute to folding and stability in this structural motif. Determining metal ion affinities and influence of metals on folding and catalysis in the hairpin ribozyme. Developing methods for site-specific EPR-active spin labels to study dynamics and distances in RNA, with application to metal-dependent folding pathways in the hammerhead and Group I Intron P4-P6 subdomain. Investigating the influence of metal ions on folding and activity in a U2-U6 RNA model system for the active site of the spliceosome.